Central Hypothesis: The Hepatitis C Virus (HCV) E2 glycoprotein is a novel kinase that initiates signal transduction mechanisms modulating the following pathways: 1.) Clathrin-mediated endocytosis, through a site-specific phosphorylation of the clathrin adaptor protein-50 (AP50), a key regulator of clathrin-mediated receptor endocytosis;and 2.) Hepatocyte proliferation and liver carcinogenesis through the activation of PI3 Kinase and Akt. In pilot transaction studies and in vitro kinase assays I have obtained compelling data suggesting that E2 is a novel member of the actin-regulating kinase family (Ark/Prk kinases) that associates physically with, and phosphorylates AP50 on its phospho-acceptor Thr156, a key step for clathrin-mediated endocytosis (25,50,73). Also, we have shown that E2 is associated with AP50 in livers from HCV-infected patients, and that AP50 is phosphorylated on Thr156 to a much greater extent in these livers. In preliminary studies, we have also found that E2, in the absence of extracellular growth factors, increases PIP2, PI3K, PDK1 and Akt, as well as their activities. This signaling cascade promotes proliferation. Moreover, HCV E2 markedly stimulates hepatocyte DNA replication to an even greater extent than classic tumor promoters TGF( and EGF. 1. The physiological relevance of the interaction between HCV E2 and AP50 and the phosphorylation of AP50 in a unique HCV infection system of primary human hepatocytes. We will analyze the protein motifs of E2 which are indispensable for association with and phosphorylation of AP50 in the HCV Huh-7 infection system. 2. The effects of HCV E2 on proliferation in HCV infected primary human hepatocyte cultures and in the HCV Huh-7 infection system. These recently characterized HCV E2 mechanisms have yet to be explored in an HCV-infected normal primary human hepatocyte model system. This will be an extremely valuable model to study these intriguing HCV E2 mechanisms, and possibly others, in the presence of the entire, naturally occurring HCV viral particle with a complete life cycle, obtained directly from patients. Mutational analysis in the Huh-7 HCV infection system is necessary in order to investigate the roles of the individual motifs of E2 and their importance in HCV infection. In addition, the discovery and mechanistic studies of a novel viral kinase has extensive implications in the fields of HCV, general virology, clathrin-mediated endocytosis, and signal transduction.